Composition and use of rar antagonists for promoting chondrogenesis

ABSTRACT

The invention provides compositions comprising a RAR antagonist for promoting chondrogenesis and to methods employing such compositions for treating cartilage and associated bone abnormalities resulting from injury or disease and for ex vivo tissue engineering.

STATEMENT OF PRIORITY

The present invention is a continuation of U.S. application Ser. No.09/856,324 (allowed), having a filing date of Aug. 23, 2001, which is a35 U.S.C. § 371 national phase application of PCT Application No.PCT/CA99/01106, having an international filing date of Nov. 19, 1999,and which claims priority to Canadian Application No. 2,254,429, filedNov. 19, 1998, the entire contents of each of which are incorporated byreference herein.

FIELD OF THE INVENTION

The invention relates to compositions for promoting chondrogenesis andto methods for treating cartilage and bone abnormalities resulting frominjury or disease and for ex vivo tissue engineering.

BACKGROUND OF THE INVENTION

Retinoic acid (RA) is known to have an important signalling role in theregulation of embryonic development and cell differentiation. Thebiological effects of RA are transduced via two classes of nuclearreceptors, designated retinoic acid receptors (RARα, RARβ and RARγ) andretinoid X receptors (RXRα, RXRβ and RXRγ) (Giguere et al., (1987),Nature, 350, 624-629).

Many studies have been made of the role of RA and its receptors in theprocess of chondrogenesis and skeletal development. For example, downregulation of RARα has been stated to be important for chondrogenicexpression (Cash et al. (1997) J. Cell Biol. 136, 445-457). Downregulation of RAR-β2 using antisense oligonucleotides was found tostimulate chondrogenesis and thus it was suggested that RAR-β2 helps toprevent mesenchymal cells from expressing their chondrogenic bias.Underhill et al. (1998) Micro. Res. Tech. 43, 137-155 reported thatabnormal expression of RARs inhibits chondrogenesis. However, it wasalso found that absence of RAR can lead to deficiencies in cartilageformation while also promoting chondrogenesis at ectopic sites.

Addition of RA and RAR-specific agonists has been shown to inhibitcartilage formation in limb bud micromass cultures, and act as ateratogen in vivo to negatively affect skeletal development (Kistler(1987) Arch. Toxicol. 60, 403-414; Kochhar (1973) Teratology 7, 289-295;Kochhar and Aydelotte (1974) J. Embryol. Exp. Morph. 31, 721-734;Kwasigrich and Kochhar (1980) Anat. Embryol. 161, 105-113. In contrast,addition of retinoic acid to micromass cultures has also beendemonstrated to stimulate cartilage formation (Paulsen et al. (1994)Dev. Dynam. 201, 310-323; Paulsen et al. (1994) Dev. Biol. 30A,181-186).

Addition of an RAR antagonist completely reversed the inhibitory actionof an RAR agonist on chondrocyte differentiation in rat and mouse embryolimb bud mesenchymal cells in vitro (Eckhardt and Schmitt (1994)Toxicol. Letters, 70, 299-308; Kocchar et al. (1998) Int. J. Dev. Biol.,42, 601-608). These authors, however, found that the antagonist alonehad no effect on limb bud mesenchymal cell differentiation.

The precise role of RARs in chondrogenesis was unclear from thesestudies. Specifically, it was unclear whether RARs functioned to inhibitchondrogenesis or stimulate that process. Overall, the role of RARantagonists in the process of chondrogenesis was not clearlyestablished.

SUMMARY OF THE INVENTION

It has now been demonstrated that RAR activity alone has a fundamentalrole in controlling the transition of chondroprogenitor cells intochondrocytes. It has also now been demonstrated that administration ofRAR antagonists can initiate chondrogenesis and stimulate cartilageformation in vitro and in vivo. Furthermore, it is now also demonstratedthat administration of a RAR antagonist can rescue cartilage formation.This provides a basis for the development of therapeutic compositionsand uses of such compositions to treat disorders involving abnormalcartilage formation.

The present invention now provides therapeutic compositions and methodsfor the treatment of disorders involving abnormal cartilage formationand associated abnormal skeletal development resulting from disease ordue to trauma.

In accordance with one embodiment, the present invention provides apharmaceutical composition comprising an effective amount of an RARantagonist and, optionally, a pharmaceutically acceptable carrier.

RAR antagonists for use in the present invention are characterized byhaving a stimulating effect on cartilage formation and as a result onassociated bone development in a vertebrate. RAR antagonists may bedefined as any chemical that binds to one or more of the RAR subtypeswith a Kd of less than 1 micromolar. Conventionally, a RAR antagonist isa chemical agent that inhibits the activity of an RAR agonist.

In accordance with a further embodiment, the invention provides a methodfor stimulating cartilage formation in a vertebrate, the methodcomprising administering to the vertebrate an effective cartilageformation stimulating amount of an RAR antagonist.

In accordance with a further embodiment, the invention provides a methodfor treating damaged cartilage and associated bone in a subject,comprising administering to the subject an effective amount of an RARantagonist, wherein the RAR antagonist stimulates cartilage repair andformation which mediates associated bone repair.

In accordance with a further embodiment, the invention provides a methodfor enhancing osseous integration of orthopedic or dental implants in asubject comprising administering to the subject an effective amount ofan RAR antagonist.

In accordance with a further embodiment, the invention provides a methodfor treating arthritis in a subject, comprising administering to thesubject an effective amount of an RAR antagonist.

In accordance with a further embodiment, the invention provides a methodfor treating arthritis in a subject, comprising administering to thesubject chondrogenic cells treated with an effective amount of an RARantagonist.

According to one embodiment of the invention, there is provided acomposition for inducing chondrogenesis and associated skeletaldevelopment in a vertebrate, the composition comprising:

-   -   a RAR antagonist; and    -   a pharmaceutically acceptable carrier.

According to another embodiment of the invention, there is provided amorphogenic device for implantation at a cartilage site in a vertebrate,the device comprising:

-   -   an implantable biocompatible carrier; and    -   a RAR antagonist dispersed within or on said carrier.

According to yet another embodiment of the invention, there is providedthe use of a composition comprising a RAR antagonist and apharmaceutically acceptable carrier, for inducing chondrogenesis invitro.

According to yet another embodiment of the invention, there is provideda method for producing a chondrocyte from a chondroprogenitormesenchymal cell comprising contacting a chondroprogenitor mesenchymalcell with a RAR antagonist in vitro.

According to another embodiment of the invention, there is provided animplantable prosthetic device for repairing cartilage-associatedorthopedic defects, injuries or anomalies in a vertebrate, the devicecomprising:

-   -   a prosthetic implant having a surface region implantable        adjacent to or within a cartilage tissue.    -   a RAR antagonist composition disposed on the surface region in        an amount sufficient to promote enhanced cartilage growth into        the surface.

According to another embodiment of the invention, there is provided amethod for promoting in vivo integration of an implantable prostheticdevice into a target cartilage tissue of a vertebrate, the methodcomprising the steps of:

-   -   providing on a surface of the prosthetic device a composition        comprising a RAR antagonist and a pharmaceutically acceptable        carrier and    -   implanting the device in a vertebrate at a site where the target        cartilage tissue and the surface of the prosthetic device are        maintained at least partially in contact for a time sufficient        to permit tissue growth between the target cartilage tissue and        the device.

According to yet another embodiment of the invention, there is provideda method for promoting natural bone formation at a site of skeletalsurgery in a vertebrate, the method comprising the steps of delivering aRAR antagonist composition to the site of the skeletal surgery wherebysuch delivery indirectly promotes the formation of new bone tissuemediated by cartilage.

According to another embodiment of the invention, there is provided amethod for repairing large segmental skeletal gaps and non-unionfractures arising from trauma or surgery in a vertebrate, the methodcomprising delivering a RAR antagonist composition to the site of thesegmental skeletal gap or non-union fracture whereby such deliverypromotes the formation of cartilage which mediates new bone tissueformation.

According to yet another embodiment of the invention, there is provideda method for aiding the attachment of an implantable prosthesis to acartilage site and for maintaining the long term stability of theprosthesis in a vertebrate, the method comprising coating selectedregions of an implantable prosthesis with a RAR antagonist compositionand implanting the coated prosthesis into the cartilage site, wherebysuch implantation promotes the formation of new cartilage tissue andindirectly stimulates bone formation.

According to a further embodiment of the invention, there is provided amethod of producing cartilage at a cartilage defect site in vivo, themethod comprising:

-   -   implanting into the defect site a population of chondrogenic        cells which have been cultured in vitro in the presence of a RAR        antagonist.

According to another embodiment of the invention, there is provided amethod for treating a degenerative joint disease characterized bycartilage degeneration, the method comprising:

-   -   delivering a therapeutically effective amount of a RAR        antagonist to a disease site.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further understood from the followingdescription with reference to the Figures, in which:

FIG. 1 shows photomicrographs of transgene-expressing cells. Panels(A-C) show wild-type fore limb cultures stained with alcian blue on days2, 4 and 6. Panels (D-G) show transgenic fore limb cultures stained withmagental-gal followed by alcian blue at days 2, 4 and 6. Panel (D) showshigher magnification of day 4 transgenic fore limb cultures.Transgene-expressing cells are excluded from the cartilage nodules.Panel (H) shows day 4 transgenic hind limb cultures.Transgene-expressing cells are much more abundant compared to fore limbcultures, and hence only a few small cartilage nodules have formed. Bar:(A-C, E-H) 1.0 mm (D) 0.4 mm.

FIG. 2A shows photomicrographs of transgene-expressing cells. Panels(A-H) show whole mount in situ hybridization of wild-type and transgeniccultures carried out using probes characteristic of specific stages ofchondrogenesis. Panel (A) shows Col II expression is very abundant inwild-type cultures. Panel (B) shows Col II expression in transgeniccultures is much weaker and has a more widespread distribution. Panels(C, E, G) show wild-type cultures, N-cad, gli-1, and col I are allabundantly expressed in perinodular regions but are only weaklyexpressed in the core of the nodules. Panels (D, F, H) show that intransgenic cultures, N-cad, gli-1 and col-I are expressed throughout thecultures with no apparent downregulation as seen in the center of thenodules of wild-type cultures. Bar: (A-H) 0.4 mm.

FIG. 2B shows the results of northern blot analysis using total hindlimb bud RNA indicating that col II is downregulated in transgenic hindlimbs at E 14.5, and gli-1 is downregulated in transgenic hind limbs atE 12.5.

FIG. 3 shows transgene-expressing cells respond differently to BMP-2.Panel (A) shows quantitative analysis of nodule formation in vitro.There are fewer cartilage nodules in the fore and hind limb cultures ofuntreated transgenic cultures compared to those in untreated wild-typecultures. BMP-2 treatment increases the number of nodules in bothwild-type and transgenic cultures, however, the increase seen inwild-type cultures is more dramatic. Panels (B-D) show wild-typecultures treated with BMP-2 stained with alcian blue at days 2, 4 and 6.In response to BMP-2, there is a noticeable increase in nodule numberand in nodule size, with cartilage nodules located at the periphery ofthe cultures becoming the largest. Panels (F-H) show transgenic culturestreated with BMP-2 and stained with magenta-gal followed by alcian blueat days 2, 4 and 6. In response to BMP-2 there is an increase in nodulenumber and in nodule size. There are also many more condensations oftransgene-expressing cells. Panel (E) shows higher magnification of day4 wild-type cultures treated with BMP-2. Panel (I) shows highermagnification of day 4 transgenic cultures treated with BMP-2. BMP-2stimulates condensation of transgene-expressing cells, but they remainexcluded from cartilage nodules. Bar: (A-C, E-G) 1.0 mm (D, H) 0.4 mm.

FIG. 4 shows Col II expression is altered in transgenic cultures. Panels(A, B) show Col II expression in wild-type and transgenic culturesrespectively. In wild-type cultures, col II is abundantly expressedwithin cartilage nodules and is much weaker in transgenic cultures witha less distinct expression pattern. Panel (C) shows that in response toBMP-2 the number of cartilage nodules in wild-type cultures that expresscol II abundantly increases. Panel (D) shows that in transgenic culturestreated with BMP-2 there are a few nodules with abundant col IIexpression and several condensations with weaker expression. Bar: (A-D)0.4 mm.

FIG. 5 shows BMP-2 and AGN194301 exhibit different chondrogenicstimulatory properties. Panel (A) shows untreated wild-type culturesstained with alcian blue on day 6. Panels (B, C) show wild-type culturestreated with BMP-2 for the first two days and three days of culture,respectively, and stained with alcian blue on day 6. These culturesresemble untreated cultures. Panel (D) shows wild-type cultures treatedcontinuously with BMP-2 and stained with alcian blue on day 6. Panels(E, F) show wild-type cultures were treated after two or three daysrespectively and stained with alcian blue on day 6. These culturesresemble cultures that were treated continuously with BMP-2. Panel (G)shows untreated wild-type cultures stained with alcian blue on day 8.Panels (H, I) show wild-type cultures treated with AGN194301 for thefirst two days and three days of culture, respectively, and stained withalcian blue on day 8. These cultures resemble cultures treatedcontinuously with AGN194301 in that there are several smaller nodules.Panel (J) shows wild-type cultures were treated continuously withAGN194301 and stained with alcian blue on day 8. Panels C, L) showwild-type cultures treated after two or three days respectively andstained with alcian blue at day 8. The size and number of cartilagenodules is less compared to cultures treated continuously. Bar: (A-L)1.0 mm. Panel (M) shows quantification of cartilage nodule formation inresponse to BMP-2 and AGN194301. In response to BMP-2, or AGN194301alone, there is a dramatic increase in the number of nodules that form.When BMP-2 and AGN194301 are added together, the number of nodulesformed is greater than when each is added alone.

FIG. 6 shows the rescue of cartilage formation in Noggin-treatedcultures by addition of AGN194301. Panel (a) shows wild-type culturesstained with alcian blue on day 6. Panel (b) shows wild-type culturestreated with Noggin (10 ng/ml) and stained with alcian blue on day 6. Incomparison to control cultures, only a few weakly stained nodules areevident and these are present within the center of the culture wherecell density is highest. Panel (c) shows wild-type cultures treated with10 ng/ml Noggin and 1 μM AGN194301. When Noggin and AGN 194301 are addedtogether, there is no apparent decrease in nodule formation incomparison to untreated cultures. Similar to that observed in controlcultures, nodules are distributed throughout the culture. Panel (d)shows the quantification of cartilage nodule formation in response toNoggin and AGN194301. Treatment of Noggin alone dramatically reduced thenumber of cartilage nodules to ˜10% of that of control cultures.Addition of AGN194301 to Noggin-treated cultures rescues cartilageformation and leads to an increase in cartilage nodule formationcomparable to that of control cultures. Panels (e, f) show the analysisof col IIa and col II expression in Noggin-treated cultures,respectively, revealing a comparable number of col 2a expressingaggregates and col II expressing nodules. The bar represents 1 mm.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have demonstrated the use and effectiveness of RARantagonists to stimulate chondrogenesis and skeletal development.

Limb Bud Development

Patterning of the vertebrate limb bud relies on the cooperative actionof several signalling centres. Signals have been identified that emanatefrom the apical ectodermal ridge (AER), ventral ectoderm (VE) and aregion within the distal posterior margin termed the zone of polarizingactivity (ZPA). The AER is important in specifying proximodistal axialidentity, while the VE and ZPA are important in determiningdorsal/ventral and anteroposterior identity, respectively. Each isessential for the proper patterning and outgrowth of the limb, and theirperturbation results in defects of the paraxial skeleton. The mechanismby which these patterning cues influence mesenchyme commitment isthought to occur within the progress zone (PZ) which underlies the AER.Cells within the progress zone receive signals from all three signalingcentres, integrating them into a positional identity that is fixed asthe cells leave the PZ during limb outgrowth. Shortly thereafter thispositional identity is translated into a cell identity and thecorresponding differentiation program is initiated. With respect toskeletal formation, these signals culminate in the commitment ofmesenchymal cells to the chondrocytic lineage.

Bones within the limb are formed from a cartilage precursor and thecartilage forms from condensed mesoderm. These condensations representthe earliest stages of limb patterning and are considered to be theforbears of the mature limb bones (Ede (1983) Cellular condensations andchondrogenesis. in Cartilage: Development, Differentiation and Growth,B. K. Hall, ed. (New York: Academic Press), pp. 143-185. Followingcondensation, the mesodermal cells in the interior of each condensationdifferentiate into chondrocytes. This differentiation occurs in concertwith limb outgrowth, such that proximal mesenchymal cells (close to thebody wall) that are fated to become chondrocytes differentiate prior tomore distal cells. The spatiotemporal regulation of mesenchymedifferentiation into chondrocytes is a crucial step in endochondral boneformation in that it preserves the pattern of the bone primordiaestablished earlier in limb development and provides a suitable matrixfor subsequent ossification. Despite the importance of this stage inskeletal development, mechanisms that control mesenchyme differentiationinto chondrocytes are poorly understood.

During limb outgrowth, signals that promote as well as inhibitchondrogenesis are important determinants in limb ontogeny (Wolpert,(1990) J. Cell Sci. Suppl. 13, 199-208. Many molecules have beenidentified that promote chondrogenesis in vivo and in vitro. Some ofthese include members of the transforming growth factor (TGF)-βsuperfamily, bone morphogenetic proteins (BMPs), gdf-5, and TGF-β-1, -2and -3 (Hogan (1996) Curr. Opin. Genet. Dev. 6, 432-438; Kingsley (1994)Genes Dev. 8, 133-146; Moses and Serra (1996) Curr. Opin. Genet. Dev. 6,581-586; Wozney and Rosen (1998) Clin. Ortho. Rel. Res. 346, 26-37. Theimportance of bmp-5 and gdf-5 in limb development is illustrated by therecent mapping of the classical mouse mutations for short ear andbrachypod to their respective loci (Kingsley et al. (1992) Cell 71,399-410; Storm et al. (1994) Nature 368, 639-643). Both of these mutantsexhibit malformations in appendicular skeletal formation which, for thecase of brachypod, has been attributed to a defect in chondrogenesisDuke and Elmer (1997) J. Embryol. Exp. Morph. 42, 209-217. Micedeficient in BMP-6 or BMP-7 also present with skeletal defects includingdelay in the ossification of the sternum in bmp-6−/− animals (Sollowayet al. (1998) Dev. Genet. 22, 321-39) and polydactyly in the hind limbsof bmp-7−/− animals Luo et al. (1995) Genes Dev. 9, 2808-2820. Bmp-2 and-4 are expressed in condensing mesenchyme early in limb development thenbecome localized to the interdigital mesenchyme and perichondrium (Joneset al. (1991) Development 111, 531-542; Lyons et al. (1990) Development109, 833-844; Rosen et al. (1989) Connect Tissue Res 20, 313-319). Mouseembryos devoid of BMP-2 or BMP-4 do not survive beyond E9.5 (Winnier etal. (1995) Genes Dev. 9, 2105-2116; Zhang and Bradley (1996) Development122, 2977-2986) making it difficult to elucidate their exact roles inskeletal development. It is known, however, that exogenously added BMP-2or -4 under certain circumstances leads to overgrowth of the limbcartilages (Duprez et al. (1996) Mech. Dev. 57, 145-157; Duprez et al.(1996) Dev. Biol. 174, 448-452). Loss or gain-of-function of noggin, aninhibitor of BMP-2, -4 and -7, contributes to either overgrowth or areduction in limb cartilages, respectively (Brunet et al. (1998) Science280, 1455-1457; Capdevila and Johnson (1998) Dev Biol 197, 205-217).Together, these observations suggest an important role for the BMPs,especially BMPs-2 and -4 in formation of the cartilaginous elements ofthe limb bud.

It is well established that retinoic acid (RA), the active derivative ofvitamin A is essential for normal embryonic development. Exposure ofembryos to excess RA results in a range of defects depending not only onthe dose of RA, but also on the timing of its administration (Shenfelt(1972) Teratology 5, 103-118). For instance when administered to E 11.5to E 14.5 mouse embryos, large doses of RA cause limb defects (Kochar(1973) Teratology 7, 289-295; Kwasigroch and Kochar (1980) Anat.Embryol. 161, 105-113). This period during which RA treatment has themost dramatic effects on limb formation coincides with the timing ofchondrogenesis in the limb bud.

RA exerts most of its biological effects primarily through receptorsbelonging to the steroid hormone family of nuclear receptors. There aretwo subfamilies of nuclear retinoid receptors, the RA receptors (RARs)and the retinoid X receptors (RXRs), with three subtypes of each (α, β,and λ) (Chambon (1996) FASEB J. 10, 940-954; Mangelsdorf et al. (1994)“The retinoid receptors,” in The Retinoids: Biology, Chemistry, andMedicine, Sporn, Roberts and Goodman, eds. (New York: Raven Press Ltd.),pp. 319-349. All three subtypes of the RARs have been suggested to havesome role in chondrogenesis (Underhill and Weston, (1998) Micro. Res.Tech. 43, 137-155). The expression patterns of the RARs in thedeveloping limb are consistent with their proposed roles in skeletalformation. Between E9.5 and E11.5, RARα and λ are expressed throughoutthe limb mesenchyme (Dolle et al. (1989) Nature 342, 702-705; Ruberte etal. (1990) Development 108, 213-222. Beyond this stage, RARα isdownregulated within the cartilaginous areas, while RARλexpressionremains localized to these areas. RARα expression becomes restricted tothe interdigital region (IDR) overlapping with RARE expression at thistime, and is also present in the perichondrium. In addition to beingpresent in the IDR, RARβ is expressed in the interior, anterior, andposterior necrotic zones (Dolle et al. (1989) Nature 342, 702-705;Mendelsohn et al. (1991) Development 113, 723-734). While null mutantsof either RARα, RARβ or RARλ exhibit no limb skeletal malformations(Ghyselinck et al. (1997) Int. J. Dev. Biol. 41, 425-44; Lohnes et al.(1993) Cell 73, 643-658; Lufkin et al. (1993) Proc. Natl. Acad. Sci. USA90, 7225-7229; Luo et al. (1995) Mech. Dev. 53, 61-71), compoundhomozygous null alleles of RARα and RARλ exhibit a range of severe limbabnormalities from reductions to duplications (Lohnes et al. (1994)Development 120, 2723-2748). Thus, these results demonstrate animportant function for the RARs in skeletal development.

To further explore RAR function in limb development the presentinventors have used an existing transgenic line of mice that overexpressa weak constitutively active RARα1 (caRARα) in the developing limb bud(Cash et al. (1997) J. Cell Biol. 136, 445-457). Transgenic animalspresent with a number of limb skeletal abnormalities as a result oftransgene-mediated inhibition of cartilage formation.Transgene-expressing cells do not differentiate into chondroblasts, butinstead remain as condensed mesenchyme. Conversely, an RARα antagonistwas demonstrated to stimulate cartilage formation in wild-type limbmesenchymal cultures. Addition of BMP-2 or -4 to in vitro cultures oftransgenic mesenchyme dramatically stimulated condensation oftransgene-expressing mesenchymal cells but failed to induce chondroblastdifferentiation. More importantly, addition of this antagonist was foundto rescue chondrogenesis in noggin treated wild-type cultures. Takentogether, the results demonstrate that loss of RAR activity isnecessary, and supersedes the requirement of BMPs, for chondroblastdifferentiation during limb outgrowth.

Regulation of chondroblast differentiation is critical for the properformation of the appendicular skeleton. Herein it is now demonstratedthat RAR activity has a fundamental role in controlling the transitionof prechondrogenic cells to chondroblasts, and that the loss of RARactivity stimulates cartilage formation. The importance of RARs inmediating chondroblast differentiation is further exemplified by theobservations that addition of BMP-2 and -4 is not sufficient to rescueRARα expressing prechondrogenic cells, while an RARα specific antagonistis able to rescue cartilage formation in noggin-treated cultures.Therefore, BMPs and RARs function in a sequential manner to orchestratechondroblast differentiation during limb bud outgrowth.

Function of RARs During Skeletal Development

Overexpression of a caRARα in limb mesenchyme inhibits chondroblastdifferentiation and maintains the prechondrogenic cell phenotype. Thephenotype of cells expressing the transgene is consistent with that ofcondensed mesenchymal cells and perichondrial cells. Both of these celltypes are similar in that they are chondroprogenitors which have not yetundergone chondroblast differentiation. Condensed mesenchymal cells andperichondrial cells both have continued expression of N-cadherin, gli-1,and col-I but express col II only weakly, a pattern of expression thatis seen in transgenic cultures. The normal expression pattern of RARα inthe developing limb is consistent with its absence being essential forthe transition from a chondroprogenitor to a chondroblast. RARα isexpressed in the prechondrogenic condensations, the perichondrium, andin the interdigital region, but is downregulated in newly formedcartilaginous elements. The importance of the loss of RARα signal isfurther demonstrated by experiments in which the RARα specificantagonist AGN194301 increased cartilage nodule formation in primarycultures by 60%. Hence, the proper regulation of RARα activity isessential for normal cartilage development.

Surprisingly, RARα null mutants display no overt skeletal malformationsof the appendicular skeleton with the exception that a certainproportion (approx. 60% of animals surviving to 1-2 months of age) ofRARα null mutants have webbed digits (Lohnes et al. (1993) Development120, 2723-2748; Lufkin et al. (1993) Proc. Natl. Acad. Sci. USA 90,7225-7229. However, in micromass cultures, attenuation of RARα2expression with an anti-sense oligonucleotide to RARα2 stimulatedcartilage formation in vitro. This suggests that RARα alone is notessential for the maintenance of the prechondrogenic cell fate, but oneof the RARs may be able to substitute for this function, the most likelycandidate being RARγ. RARγ null mutants also have no obviousappendicular skeletal malformations. RARα/RARγ compound homozygousmutants, however, exhibit a number of skeletal defects includingreductions, duplications, and to a lesser extent, ectopic cartilageformation within the interdigital region (Development 120, 2723-2748.Ectopic cartilages are also observed at a number of additional sites inthese animals including the meninges, peritoneum, diaphragm, andsemi-lunar cusps of the heart (Lohnes et al. (1994) Development 120,2723-2748; Mendelsohn et al. (1994) Development 113, 723-734). Inaddition, the phalangeal joints of these animals are malformed. WhileRARα/β and RARβ/γ double knockouts exhibit some cartilaginousdeficiencies, they do not present with any appendicular skeletaldefects. More importantly, the skeletal defects in the RARα/γ doubleknockouts can be rescued, for the most part, by the single allele ofRARα2.

Results from knockout studies combined with the in vivo expressionpatterns of RARs suggest that both RARα and RARγ are important inregulating chondroblast differentiation in the limb. RARβ knockoutsexhibit no skeletal abnormalities, and RARβ mRNA is absent inprecartilaginous condensations during limb ontogeny (Ghyselnick et al.(1997) Int. J. Dev. Biol. 41, 425-447). Thus, the combined activity ofRARα and RARγ, but not RARβ is likely involved in regulating cartilagedifferentiation in the limb bud. In other areas of the developingembryo, chondrogenesis may also rely on the actions of specificcombinations of RARs.

Sequential Action of BMPs and RARs in Skeletal Development

The BMPs have been shown to be important in many aspects of endochondralbone formation including the commitment and differentiation ofmesenchymal cells to the chondrocytic lineage. During limb outgrowthbmp-2/-4 are expressed within the condensing mesenchyme, theperichondrium and the interdigital region (Jones et al. (1991)Development 111, 531-542; Lyons et al. (1990) Development 109, 833-844;Rosen et al. (1996) Ann. N.Y. Acad. Sci. 785, 59-69. Unfortunately, nullmutants have not been informative in sorting out the function ofBMPs-2/-4 in these regions (Winnier et al. (1995) Genes Dev. 9,2105-2116; Zhang and Bradley (1996) Development 122, 2977-2986.Additional studies, utilizing dominant-negative or constitutively activeBMP type II receptors in vitro and in vivo have demonstrated, however,that BMP signaling (most likely BMP-2 and 4) is a requisite step incartilage formation (Zou et al. (1997) Genes Dev. 11, 2191-2203). Theseobservations have been complemented by experiments in which BMPs wereoverexpressed in the developing chick limb. It has been demonstratedthat the BMPs can stimulate cartilage formation (Duprez et al. (1996)Mech. Dev. 57, 145-157; Duprez et al. (1996) Dev. Biol. 174, 448-452)and modify skeletal element patterning in addition to stimulatingapoptosis within the interdigital region (Macias et al. (1997)Development 124, 1109-1117; Yokouchi et al. (1996) Development 122,3725-3734). Furthermore, loss or gain of function studies with noggin,an inhibitor of BMP-2 and -4 with lower affinity for BMP-7, has shownthat BMP-2 and -4 are important in skeletal development and thatregulation of BMP signaling is required for delineation of the variousskeletal elements (Brunet et al. (1998) Science 280, 1455-1457;Capdevila and Johnson (1998) Dev Biol 197, 205-217). As describedherein, exogenously added noggin inhibits cartilage formation inmicromass cultures. BMP-2 has been shown to stimulate the commitment anddifferentiation of pluripotent mesenchymal cells to the chondrocyticlineage (Ahrens et al. (1993) DNA Cell Biol. 12, 871-880; Wang et al.(1993) Growth Factors 9, 57-71). Thus, BMPs, especially -2/-4, areimportant in early skeletal development, and appear to have propertiesconsistent with a functional importance in commitment anddifferentiation of mesenchymal cells to chondrocytes.

Overexpression of a caRARα prevents prechondrogenic celldifferentiation, even in the presence of BMP-2 or -4. Both BMP-2 and -4enhance the expression of the prechondrogenic phenotype by stimulatingthe formation of numerous condensations as confirmed by magenta-galstaining and by in situ hybridization with probes for N-cadherin, gli-1,col-I and col-II. Nonetheless, neither BMP-2 nor BMP-4 was able toinduce chondroblast differentiation of transgene-expressing cells. Theseresults demonstrate that the regulation of RAR activity operatesdownstream of BMP-signaling in the chondroblast differentiationsequence. Additional evidence to suggest this comes from theobservations that inhibition of cartilage formation by noggin can becircumvented by treatment of cultures with an RAR antagonist. Duringparaxial development, RARα, RARγ, are co-expressed in the condensingmesenchyme and in the perichondrium, which are both targets of BMP-2 and-4. Hence, these results suggest that during limb outgrowth BMP-2 and -4are important in stimulating prechondrogenic cell condensation, whereasa loss of RARα activity is important in allowing the differentiation ofthese cells. In this manner, the BMP and RA-signaling pathways mayfunction sequentially in the commitment and differentiation ofmesenchymal cells, respectively, during limb outgrowth.

RAR Function in Chondroblast Differentiation: Conservation in OtherDevelopmental Processes

A similar hierarchy of BMP and RA signaling may be operating in otherdevelopmental processes. BMPs-2 and -4 and RARs are co-expressed in anumber of different regions within the developing embryo. As mentionedpreviously, BMP-2 and -4 are co-expressed with RARα, β, and γ in theinterdigital region. Application of BMPs to the interdigital regionenhances interdigital apoptosis (Macias et al. (1997) Development 124,1109-1117; Yokouchi et al. (1996) Development 122, 3725-3734), whileoverexpression of a dominant-negative BMPIIR inhibits interdigitalapoptosis and webbed digits (Zou and Niswander (1996) Science 272,738-741; Zou et al. (1997) Genes Dev. 11, 2191-2203). In addition, lossof RARα, or loss of β and γ together, leads to a cessation ofinterdigital apoptosis and webbed digits. When cultured prior to theinitiation of apoptosis, explants of the interdigital region will formcartilage unless RA is present in which case they will undergo apoptosis(Lee et al. (1994) Dev. Dynam. 201, 297-309). Similarly, addition of RAto in vitro cultured limbs stimulates regression of the interdigitalregions (Lussier et al. (1993) Int. J. Dev. Biol. 37, 555-564).Together, these observations suggest that the BMPs and RA-signalingpathways coordinate interdigital cell death, in addition tochondrogenesis.

During heart formation BMP-2 and -4 appear to be important in specifyingventricular cardiomyocytes whereas the RARs regulate the differentiationof this population of cells. Loss of RAR activity through inactivationof RXRα, RXRβ, RARα causes precocious differentiation of ventricularmyocytes (Kastner et al. (1997) Development 124, 4749-4758). Hence, RARsare functioning in the heart to regulate progenitor cell differentiationas they do within the limb.

One of the functions of RARs in limb development is to regulate thedifferentiation of skeletal progenitor cells. In this manner, RARactivity may specify the size of progenitor cell populations, and/orinfluence cell fate decisions by modulating the competency of cells torespond to inductive signals, such as BMPs. The status of cellular RARactivity, therefore, appears to be an important determinant in thespatiotemporal regulation of cell differentiation in the developinglimb, heart and spinal cord.

RAR Antagonists

With the demonstration that RAR antagonist compounds can act as potentstimulators of chondrogenesis and associated skeletal development,various therapeutic in vivo and in vitro uses of such antagonists arenow made possible especially those uses involving abnormalchondrogenesis and related skeletal development.

The RAR antagonist AGN 194301, shown in Table 1, has been demonstratedto stimulate cartilage formation and promote chondroblastdifferentiation. AGN 194301(2-Fluoro-4-[(1-(8-bromo-2,2-dimethyl-4-(4-methylphenyl)-2-H-chromen-6-yl)-methanoyl)-amino]-benzoicacid) is a potent antagonist of RARα, with a high affinity for thatreceptor. It has a lower affinity for RARβ and RARγ, but does also actas an antagonist of these receptors.

In accordance with one embodiment of the invention,chondrogenesis-stimulating RAR antagonists comprise antagonist compoundswhich are highly effective against RARα and also antagonise RARβ andRARγ. Thus, the present invention encompasses RAR antagonists in generaland any agent which demonstrates RAR antagonist activity. Those ofordinary skill in the art are able to screen candidate compounds toidentify compounds having such an RAR antagonist profile by methodsavailable in the scientific literature, for example as described in Tenget al. (1997) J. Med. Chem. 40, 2445-2451.

In accordance with one embodiment of the invention,chondrogenesis-stimulating RAR antagonists comprise mono- or di-fluorosubstituted methylchromenes such as AGN 194301. The RAR antagonistcompounds of the invention may be synthesized by conventional chemicalsynthetic methods. For example, AGN 194301 may be synthesised asdescribed in Teng et al. (supra). In some cases, suitable RARantagonists may be purchased.

One skilled in the art would readily understand that several differenttypes of RAR antagonists other than those described specifically hereinare suitable for use in the present invention. Other suitable RARantagonists are taught for example in WO 9933821, WO 9924415, U.S. Pat.No. 5,877,207 and JP 10114757. Such antagonist agents include but arenot limited to AGN 193109, AGN 190121, AGN 194574, AGN 193174, AGN193639, AGN 193676, AGN 193644, SRI 11335, Ro 41-5253, Ro 40-6055, CD2366, BMS 185411, BMS 189453, CD-2665, CD 2019, CD 2781, CD 2665, CD271. Other suitable RAR antagonists for use in the present inventioninclude those disclosed in Kaneko et al. (1991) Med. Chem. Res. 1,220-225; Apfel et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89,7129-7133; Eyrolles et al. (1994) J. Med. Chem. 37, 1508-1517; Yoshimuraet al. (1995) J. Med. Chem. 38, 3163-3173; Eckharat and Schmitt (1994)Toxicol. Lett. 70, 299-308; and Teng et al. (1997) J. Med. Chem. 40,2445-2451.

The chondrogenesis-stimulating RAR antagonists of the invention areuseful for the treatment and management of skeletal problems orabnormalities resulting from disease or trauma in vertebrates, includinghumans and other mammals, including horses. They may be used in severaltherapeutic applications where increased chondrogenesis is desired andalso for stimulation of skeletal development.

Therapeutic applications of these antagonists include the stimulation ofnew cartilage formation and accelerate associated bone repair throughendochondral ossification.

A pharmaceutical composition comprising at least onechondrogenesis-stimulating RAR antagonist may be applied locally to afracture site, for example by means of a biodegradable sponge, gel,coating or paste. A suitable gel for use would be a collagen type gelsuch as collagen I.

The antagonists of the present invention may also be used for thetreatment of orthopedic or dental implants to enhance or accelerateosseous integration. A pharmaceutical composition comprising at leastone chondrogenesis-stimulating RAR antagonist may be directly appliedlocally to the site of desired osseous integration or alternatively as acoating on implants.

The RAR antagonists may also be used for promoting in vivo integrationof implantable prosthetic devices. In general, the RAR antagonistcompositions of the invention may be applied to synthetic bone graftsfor implantation whereby the antagonist composition stimulates cartilageformation and indirectly bone formation. The compositions thus havenumerous applications in the orthopedic industry. In particular, thereare applications in the fields of trauma repair, spinal fusion,reconstructive surgery, maxillo-facial surgery and dental surgery. Theability of the RAR antagonist compositions to stimulate local naturalbone growth provides stability and rapid integration, while the body'snormal cell-based bone remodeling process slowly resorbs and replaces aselected implant with natural bone. Implants suitable for in vivo useare generally known to those skilled in the art.

The RAR antagonists of the invention may be used for cartilage andskeletal reconstruction. In such an application, the antagonists can beused for ex vivo tissue engineering of cartilage or skeletal tissue forimplantation in a vertebrate. Cells can be treated with a RAR antagonistduring osteochondral autograft or allograft transplantations (Minas etal. (1997) Orthopedics 20, 525-538). In autograft transplantations,chondrogenic cells or cells with chondrogenic potential are removed froma patient (e.g. from a rib) and used to fill a cartilaginous lesion. Analternative method involves expanding these cells in vitro, thenimplanting them into a cartilaginous lesion. A pharmaceuticalcomposition comprising at least one chondrogenesis-stimulating RARantagonist would be used to treat the cells in in vitro culture prior toengraftment and/or after engraftment through intra-articular injection.The use of the RAR compositions of the invention may eliminate the painand costs associated with the bone harvest procedure required inautograft transplants. Furthermore, the RAR compositions can be madesynthetically thus reducing the possibility of transmission of infectionand disease, as well as diminishing the likelihood of immunologicalrejection by the patient.

The antagonist compositions of the present invention may also be usedfor the treatment of arthritis, either osteoarthritis or other types ofarthritis including rheumatoid arthritis. To reverse or slowdegenerative joint disease characterized by cartilage degeneration, apharmaceutical composition comprising at least onechondrogenesis-stimulating RAR antagonist would be applied locallythrough intra-articular injection or in combination with aviscosupplement. The composition could be provided in either afast-release or slow-release formulation. Such compositions have use inpatients with degenerative hip or knee joints, for example.

In general, the RAR antagonists may be used to stimulate in vitrochondrogenesis from mesenchymal precursor cells and in vitro formationof chondrocytes. Such cell culture materials and methods are known tothose skilled in the art and are also described herein in the examples.Cells and tissues treated with a selected RAR antagonist in vitro can beused therapeutically in vivo or alternatively for in vitro cellularassay systems.

The pharmaceutical compositions of the invention may be used incombination with other chondrogenic stimulators, e.g. bone morphogeneticproteins (BMPs) especially BMP-2 and BMP-4, osteogenic proteins (OPs)such as OP-1 and/or cytokines to enhance and/or maintain the effects ofthe compositions. Both BMPs and OPs are proteins belonging to the TGF-βsuperfamily which represent proteins involved in growth anddifferentiation as well as tissue morphogenesis and repair. It is alsounderstood that the RAR antagonist compositions of the invention mayadditionally comprise other chondroinductive agents or factors, definedas any natural or synthetic organic or inorganic chemical or biochemicalcompound, or mixture of compounds which stimulate chondrogenesis. It isfurther understood that the RAR antagonist compositions of the inventionmay also comprise other growth factors known to have a stimulatoryeffect on cartilage growth and formation.

The RAR antagonist compositions described herein appear to have a moredramatic effect on chondrocyte function than the BMPs and showlonger-lived effects. The RAR antagonists are also more cost effectiveto manufacture than BMPs.

Those of ordinary skill in the art are familiar with various methods offormulating pharmaceutical compositions for local administration indiseases such as arthritis. For example, Adams et al. (1995)Osteoarthritis & Cartilage 3, 213-225, describes viscosupplementation inosteoarthritis; Wozney and Rosen (1998) Clin. Ortho. Rel. Res. 346,26-37, describes delivery methods used for BMPs to effect bone repairand formation. These formulation methods may be employed to prepare theRAR compositions of the invention.

For therapeutic applications in accordance with the present inventionthe RAR antagonists are incorporated into pharmaceutical compositionsformulated for oral or parenteral administration, the latter routeincluding intravenous and subcutaneous administration. Parenteraladministration may be by continuous infusion over a selected period oftime. As such, the compositions may be provided as tablets, pills,capsules, solutions, suspensions, creams, gels, and the like.

An RAR antagonist may be orally administered with an inert diluent orwith an assimilable edible carrier, or it may be enclosed in hard orsoft shell gelatin capsules, compressed into tablets or incorporateddirectly with the food of the diet. For oral therapeutic administration,the RAR antagonist may be incorporated with excipient and used in theform in ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like.

In one embodiment of the present invention, a pharmaceutical compositionfor administration to subjects in a biologically compatible formsuitable for administration in vivo for treating abnormalchrondrogenesis and associated skeletal development comprises a safe andeffective amount of a selected RAR antagonist alone, or in combinationwith other agents and/or pharmaceutically acceptable carriers. Thecomposition may be administered to any living organism including humansand animals. By “safe and effective amount” as used herein is meantproviding sufficient potency in order to decrease, prevent, ameliorateor treat a chondrogenesis or skeletal disorder affecting a subject whileavoiding serious side effects. A safe and effective amount will varydepending on the age of the subject, the physical condition of thesubject being treated, the severity of the disorder, the duration oftreatment and the nature of any concurrent therapy. Dosage regima may beadjusted to provide the optimum therapeutic response. For example,several divided doses may be administered daily or the dose may beproportionally reduced as indicated by the exigencies of the therapeuticsituation. The compositions are preferably in the form of a unit doseand will usually be administered as a dose regimen that depends on theparticular tissue treatment.

The compositions described herein can be prepared by known methods forthe preparation of pharmaceutically acceptable compositions which can beadministered to subjects, such that an effective quantity of the activesubstance is combined in a mixture with a pharmaceutically acceptablecarrier. By pharmaceutically acceptable carrier as used herein is meantone or more compatible conventional solid or liquid delivery systems asare well known in the art. Some examples of pharmaceutically acceptablecarriers are sugars, starches, cellulose and its derivatives, powderedtragacanth, malt, gelatin, collagen, talc, stearic acids, magnesiumstearate, calcium sulfate, vegetable oils, polyols, agar, alginic acids,pyrogen-free water, isotonic saline, phosphate buffer, and othersuitable non-toxic substances and medicinal agents used inpharmaceutical formulations. Other excipients such as wetting agents andlubricants, tableting agents, stabilizers, anti-oxidants andpreservatives are also contemplated. Suitable carriers are furtherdescribed for example in Remington's Pharmaceutical Sciences(Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa., USA 1985). On this basis the compositions include, albeit notexclusively, solutions of the substance in association with one or morepharmaceutically acceptable vehicles or diluents, and contained inbuffered solutions with a suitable pH and iso-osmotic with thephysiological fluids.

The RAR antagonist compositions of the invention can be provided as aliquid for local administration at a desired tissue site such as byinjection. Alternatively, the compositions of the invention can beprovided encapsulated for administration to a desired tissue site. Inone embodiment, the RAR antagonist composition may comprise at least oneRAR antagonist which may be provided as a solution or emulsion containedwithin phospholipid vesicles called liposomes. The liposomes may beunilamellar or multilamellar and are formed of constituents selectedfrom phosphatidylcholine, dipalmitoylphosphatidylcholine, cholesterol,phosphatidylethanolamine, phosphatidylserine,demyristoylphosphatidylcholine and combinations thereof. Themultilamellar liposomes comprise multilamellar vesicles of similarcomposition to unilamellar vesicles, but are prepared so as to result ina plurality of compartments in which the silver component in solution oremulsion is entrapped. Additionally, other adjuvants and modifiers maybe included in the liposomal formulation such as polyethyleneglycol, orother materials.

It is understood by those skilled in the art that any number of liposomebilayer compositions can be used in the composition of the presentinvention. Liposomes may be prepared by a variety of known methods suchas those disclosed in U.S. Pat. No. 4,235,871 and in RRC, Liposomes: APractical Approach. IRL Press, Oxford, 1990, pages 33-101.

The liposomes containing the RAR antagonist may have modifications suchas having non-polymer molecules bound to the exterior of the liposomesuch as haptens, enzymes, antibodies or antibody fragments, cytokinesand hormones and other small proteins, polypeptides or non-proteinmolecules which confer a desired enzymatic or surface recognitionfeature to the liposome. Surface molecules which preferentially targetthe liposome to specific organs or cell types include for exampleantibodies which target the liposomes to cells bearing specificantigens. Techniques for coupling such molecules are well known to thoseskilled in the art (see for example U.S. Pat. No. 4,762,915 thedisclosure of which is incorporated herein by reference). Alternatively,or in conjunction, one skilled in the art would understand that anynumber of lipids bearing a positive or negative net charge may be usedto alter the surface charge or surface charge density of the liposomemembrane.

The liposomes can also incorporate thermal sensitive or pH sensitivelipids as a component of the lipid bilayer to provide controlleddegradation of the lipid vesicle membrane.

For systemic application by intravenous delivery, it may be beneficialto encapsulate the RAR antagonist within sterically-stabilized liposomeswhich exhibit prolonged circulation time in blood. The stericallystabilized liposomes are produced containing polyethylene glycol as anessential component of their surface and the method of making suchliposomes is known to those skilled in the art.

The size of the liposomes can be selected based on the intended targetand route of administration. Liposomes of between about 10 nm to 300 nmmay be suitable. Furthermore, the composition of the present inventionmay include liposomes of different sizes.

While the composition of the present invention may be encapsulated foradministration by liposomes, it is understood by those skilled in theart that other types of encapsulants may also be used to encapsulate theRAR antagonist. Microspheres including but not limited to those composedof ion-exchange resins, crystalline ceramics, biocompatible glass, latexand dispersed particles are suitable for use in the present invention.Similarly, nanospheres and other lipid, polymer or protein materials canalso be used.

The RAR antagonist compositions of the present invention may comprise aRAR antagonist dispersed in an implantable biocompatible carrier thatfunctions as a suitable delivery or support system for the antagonist.Suitable examples of biocompatible sustained release carriers includesemi-permeable polymer matrices in the form of shaped implantablearticles such as polylactides, copolymers of L-glutamic acid,ethyl-L-glutamate, poly(2-hydroyethyl-methacrylate) or ethylene vinylacetate. Such matrices can be fabricated to have the RAR antagonistincorporated therein and be of a selected pore size to permitchondroprogenitor cells and skeletal progenitor cells to migrate within.The selected carrier material may also comprise a biodegradable,synthetic or synthetic-organic matrix such as hydroxyapatite, collagen,tricalcium phosphate or various copolymers of glycolid, lactic andbutyric acid.

The RAR antagonist composition of the present invention may also be usedwith demineralized allogenic bone and demineralized xenogenic boneoptionally treated with fibril modifying agents. Furthermore, thecomposition may be provided with a mechanical or suitable physicaldevice, influence or force such that it functions to promotechondrogenesis and skeletal development either in vitro or in vivo.

In summary, RAR antagonists have important clinical therapeutic uses fortreatment of cartilage and associated bone development defects. The RARantagonists can be used to provide such treatment both in vitro and invivo to treat a variety of conditions as a result of trauma, geneticdisease or degenerative disease negatively affecting cartilage andassociated bone development and maintenance.

EXAMPLES

The examples are described for the purposes of illustration and are notintended to limit the scope of the invention.

Methods of chemistry, protein and peptide biochemistry, cell biology,molecular biology and immunology referred to but not explicitlydescribed in this disclosure and examples are reported in the scientificliterature and are well known to those skilled in the art.

Methods Preparation of Micromass Cultures

Micromass cultures were prepared from murine E11.25 to E11.75 fore andhind limb buds as previously described with the following modifications(Cash et al. (1997) J. Cell Biol. 136, 445-457). After proteolyticdigestion cells were filtered through a Cell Sieve (40 μM, Falcon) toobtain a single cell suspension. Culture media (40% Dulbecco's modifiedEagle's medium, 60% F12 was supplemented with fetal bovine serum to 10%,Gibco-BRL) was changed daily. BMP-2 or -4 (Genetics Institutes), AGN194301 (Allergan Pharmaceuticals) and/or purified Xenopus noggin proteinwas added to culture media at a concentration of 10 ng/ml, 1 μM and 10ng/ml, respectively. Addition/removal experiments included either addingor removing supplemented media on the indicated culture day, 24 hoursafter culture initiation was considered day 1. To detecttransgene-expressing cells, cultures were fixed stained as previouslydescribed, with magenta-gal (BioSynth International Inc.) beingsubstituted for X-gal. This was followed by alcian blue staining forcartilage-specific glycosaminoglycans. Alcian blue staining ofmagenta-gal stained cultures turned the red precipitate to a purplecolor, this is a result of incubating magenta-gal stained cells at pH 1.This double-staining technique enables transgene-expressing cells to belocalized with respect to alcian blue stained cartilage nodules.

Synthesis of Riboprobes

Riboprobes were synthesized in the presence of UTP-digoxigenin with theappropriate RNA polymerase and linearized template DNA according to themanufacturers directions (Boehringer Mannheim Corp., Indianapolis,Ind.). Riboprobe complementary to collagen type II gene, was generatedfrom Bam H1 linearized pBluescript containing 1.1 kb of the collagentype II gene containing the C-propeptide and transcribed in vitro withT7 RNA polymerase. Gli-1 riboprobe was transcribed from Not 1 linearizedpBluescript containing a 1.6 kb fragment representing most of the zincfinger domain of gli-1. A 553 bp fragment of murine collagen type I(Phillips et al. (1992) Genomics 13, 1345-1346) was subcloned into pKSII (Stratagene), this was linearized with XhoI and transcribed with T7RNA polymerase. A Hind III (position 605)-BamH1 (position 1252) fragmentfrom the mouse N-cadherin cDNA was subcloned into pKSII. This constructwas linearized with Bam H1 and riboprobe synthesized with T7 RNApolymerase. For controls, sense riboprobes were synthesized from theaforementioned plasmids.

Whole-Mount in situ Hybridization of Micromass Cultures

In situ hybridizations were carried out on micromass cultures using atechnique described previously (Cash et al. (1997) J. Cell Biol. 136,445-457), with minor modifications. After permeabilization using 10μg/ml proteinase-K in phosphate buffered saline (PBS) supplemented with0.05% Triton X-100, cells were post-fixed in 4% paraformaldehyde and 2%glutaraldehyde in PBS and hybridizations were carried out at 60° C.instead of 55° C.

Transient Transfection Analysis

The ability of AGN194301 to inhibit all-trans RA induction of an RAREcontaining luciferase construct was performed in P19 embryonal carcinomacells as previously described with some modification (Underhill et al.(1994) Mol. Endo. 8, 274-285). P19 cells were seeded at a density of1.5×10⁴ cells/well in 6 well plates. Cells were transfected using thecalcium phosphate precipitation method with each well receiving 3.9 μgDNA (1.25 μg pW1RAREtk-lucif, 0.33 μg pW1ActRARα/β/β, 0.67 μgpW1Actp-galactosidase and 1.65 μg pGEM9zf(−)). Following transfectioncells were washed and fresh media was added that contained 1×10⁻⁷ Mall-trans RA and various amounts of AGN194301. Twenty-four hours latercell extracts were prepared, and luciferase and β-galactosidase activitymeasured. Luciferase activity was normalized with β-galactosidaseactivity to control for differences in transfection efficiency.

Northern Blot Analysis

Total RNA was isolated dissected and pooled limb buds from wild-type andtransgenic embryos at various gestational stages with TriPure IsolationReagent (Boehringer Manheim). RNA samples were separated byelectrophoresis of 15 μg aliquots in a 1% agarose-formaldehyde gel. RNAwas then transferred to a Hybond-N nylon membrane (Amersham LifeScience) and cross-linked by UV irradiation. Blots were pre-hybridizedin Church's Buffer (7% SDS, 0.5 M NaPi pH 7.2, 1 mM EDTA, 1% BSA) at 65°C. for at least 30 min. Radiolabeled DNA probes were synthesized byrandom priming using standard methods with the appropriate cDNA insertfragments. Hybridizations were carried out overnight at 60° C. Followinghybridization blots were washed with wash buffer (250 mM NaPi, 10% SDS)three times for 15 min. at 65° C., and exposed to BioMax X-ray film at−80° C. for 1-4 days:

Example 1 Transgene-Expressing Cells do not Contribute to CartilageNodule Formation

To further demonstrate the role of the RARs, specifically RARα, duringcartilage formation, we used a previously described transgenic mousemodel (Cash et al. (1997) J. Cell Biol. 136, 445-457). Overexpression ofa weak constitutively active RARα during limb development leads tovarious congenital malformations of the limb that are reminiscent ofthose observed in RA teratogenicity. During chondroblastdifferentiation, RARα expression is down-regulated. The continuedexpression of RARα inhibits chondroblast differentiation leading to acessation of cartilage formation and to skeletal deficiencies asobserved in the transgenic mice. Limb mesenchyme from embryonic age (E)11.5 transgenic embryos was used to set up micromass cultures to examinethe cell fate of transgene-expressing cells during in vitrochondrogenesis. Shown in FIG. 1 is a time-course of cartilage noduleformation (day-2, day-4 and day-6) in wild-type (FIG. 1A, B, and C) andtransgenic fore limb cultures (FIGS. 1E, 1F, 1G) and hind limb cultures(FIG. 1H, day-4 shown). Consistent with previous observations there aremany fewer alcian blue-stained nodules in the transgenic derivedcultures than in the wild-type cultures. Using a combination ofmagenta-gal and alcian blue staining, we clearly show thattransgene-expressing cells are, for the most part, excluded from thecartilage nodules (FIG. 1D) but appear to form condensations.Transgene-expressing cells fail to differentiate into chondroblasts incontrast to both non-transgene-expressing cells within the samecultures, and cells within wild-type cultures.

Example 2 Transgene-Expressing Cells have a Prechondrogenic Phenotype

Cartilage formation involves two well described steps: 1) condensationof mesenchymal cells; 2) differentiation of condensed mesenchyme tomatrix-producing chondrocytes (Hall and Miyake (1992) Anat. Embryol.186, 107-124). These two stages can be distinguished based onhistological staining, however, using molecular markers is generally amore reliable method for determining the phenotype of cells at eitherstage. Previous studies have shown that condensed prechondrogenic cellsexpress col II weakly and express N-cad, col I and gli-1 abundantly(Hall and Miyake (1995) Int. J. Dev. Biol. 39, 881-893; Marigo et al.(1996) Dev. Biol. 180, 273-283; Oberlender and Tuan (1994) Cell AdhesCommun 2, 521-537. Upon differentiation of these cells, col IIexpression becomes much stronger, whereas expression of N-cad, col I,and gli-1 are down-regulated. To further characterize the phenotype ofthe transgene-expressing cells, whole mount in situ hybridization wascarried out to examine the expression patterns of col II, N-cad, col Iand gli-1 in transgenic and wild-type cultures. In wild-type cultures,col II expression was very strong and localized primarily to the core ofthe cartilage nodules with weaker expression in regions surrounding thenodules (FIG. 2A). Expression of N-cad, col I and gli-1 in wild-typecultures was restricted to perinodular regions in condensed mesenchymeand was weakly expressed in the centre of the nodules (FIG. 2C, E andG). The expression patterns of these genes in the wild-type cultures isconsistent with their expression in vivo. In transgenic cultures,however, their expression patterns would indicate that whilecondensations are present the condensed cells have not yetdifferentiated. There was no downregulation of N-cad, col I or gli-1,instead they were expressed throughout the condensations (FIG. 2D, F andH), whereas col II was only weakly expressed (FIG. 2B), resembling apattern one would expect to see in condensations but not in cartilagenodules. These observations demonstrate that the transgene-expressingcells condense but do not undergo chondroblast differentiation.

Comparison of the distribution of col II expression in transgenic andwild-type cultures suggests that the transgenic cultures form a similarnumber of condensations as observed in wild-type cultures (FIG. 2A, B).Furthermore, the col II stained regions in transgenic and wild-typecultures are similar in size indicating that transgene-mediatedinhibition of chondroblast differentiation was not a consequence ofinsufficient numbers of prechondrogenic cells (Hall and Miyake (1992)Anat. Embryol. 186, 107-124). Moreover, during the culture period thetransgene-expressing cell aggregates continue to expand in size andstaining intensity (FIGS. 1E, 1F and 1G). Northern blot analysis hasconfirmed differential expression of col II and gli-1 between the hindlimbs of wild-type and transgenic mice. By E 14.5, col II isdownregulated in transgenic hind limbs (FIG. 21), whereas gli-1 isdownregulated in transgenic hind limb at E 12.5 (FIG. 2I). The in situhybridization results combined with results from Northern blot analysisdemonstrate that the phenotype displayed by transgene-expressing cellsis consistent with condensed prechondrogenic cells.

Example 3 Transgene-Expressing Cells are Refractile to BMP-StimulatedChondroblast Differentiation

To further investigate the mechanism of transgene-mediated inhibition ofchondroblast differentiation, the expression of genes known to beimportant in chondrogenesis, namely the BMPs was examined. Based ontheir expression in limb development and their well defined chondrogenicstimulatory properties, the analysis was focused on bmps-2 and -4.Hence, one plausible explanation for the transgenic phenotype was eitherthe reduced expression of bmps or overexpression of noggin which encodesan inhibitor of BMP signaling. Northern analysis using mRNA from hindlimbs of transgenic and wild-type animals at E11.5, 12.5, and E 14.5demonstrated that neither bmp2 nor noggin is differentially expressed intransgenic animals in comparison to wild-type animals (data not shown).The defect in transgenic limb mesenchyme therefore is not likely aresult of changes in expression of either of these two transcripts.However, these results do not preclude the possibility thattransgene-expressing mesenchymal cells are unresponsive to BMPs.

To evaluate whether transgene-expressing cells are able to respond toBMPs, transgenic and wild-type micromass cultures were treated with 10ng/ml BMP-2 and -4. It was demonstrated that addition of BMP-2 (FIG. 3A)and -4 dramatically increases the number of cartilage nodules inwild-type and transgenic cultures. BMP-2 treatment for six daysincreased the number of cartilage nodules by ˜125% and ˜115% inwild-type and transgenic fore limb cultures, respectively (FIG. 3A-I).Hence, addition of BMP-2 or -4 appears to rescue the chondrogenic defectpresent in transgenic cultures. To confirm this, BMP-2 treated cultureswere stained with magenta-gal followed by alcian blue (FIG. 3F, G andH). As observed in non-treated transgenic cultures, few if anytransgene-expressing cells were found to be present within the alcianblue stained cartilage nodules (FIG. 31). Addition of BMP-2, however,stimulated condensation of transgene-expressing cells (FIG. 3F-I). Thiswas confirmed by in situ hybridization, BMP-2 addition stimulated theformation of col II expressing nodules and weak col II condensations inwild-type and transgenic cultures, respectively (FIG. 4). Condensationswere observed as early as 2 days in culture and were still evident after6 days in culture. Thus, although BMP-2 or -4 inducestransgene-expressing cells to form precartilaginous condensations theyare not sufficient to overcome transgene-mediated inhibition ofprechondrogenic cell differentiation.

Example 4 Loss of Retinoic Acid Receptor Activity StimulatesChondrogenesis

It has presently been demonstrated that the continued expression of RARαinhibits the transition of prechondrogenic cells to chondroblasts. Itwas then demonstrated that the abrogation of RARα activity stimulatedchondroblast differentiation and/or cartilage formation. RAR activity asused herein refers to the level of RA-induced transcriptional activityof the RARs. Micromass cultures were treated with the RARα-specificantagonist AGN194301 to examine whether inhibition of RARα activitystimulated cartilage formation. Addition of 1 μM AGN194301 to wild-typemicromass cultures lead to a dramatic increase in nodule number with noapparent increase in nodule size as compared to untreated controlcultures (FIG. 5G, J, M). After 8 days in culture there were 60% morenodules in antagonist treated cultures than in untreated cultures (FIG.5M). Similar to the inhibition of cartilage nodule formation observed inresponse to all-trans RA treatment of micromass cultures, addition ofthe RARα-specific agonist AGN 193836 at 1 μM, decreased the number ofcartilage nodules that formed by 35% with the nodules staining onlyweakly with alcian blue. To confirm that the concentrations utilized inthese experiments inhibited RARα activity specifically, the level ofRARα, RARβ and RARγ mediated RARE activation was measured with 100 nMall-trans RA in the presence of various concentrations of antagonist. At1 μM of AGN194301, RARα signaling was inhibited to ˜0.3% of controls,while RARE and RARγ were inhibited to ˜18% and ˜26% of controls,respectively. Hence, most of the chondrogenic stimulatory properties ofthe antagonist appear to be mediated through inhibition of RARα,however, it cannot be entirely discounted that diminution of RARE orRARγ signaling may have contributed to these results. Nonetheless, lossof RAR activity stimulates cartilage formation while increased RARactivity inhibits cartilage formation.

Example 5 The RAR Antagonist and Bmp-2 have Different ChondrogenicStimulatory Properties

The loss of RAR activity and addition of BMP-2 both stimulate cartilageformation. In transgenic cultures, BMP-2 stimulated condensation but notdifferentiation of transgene-expressing cells, whereas a loss of RARαactivity induced cartilage formation. These results suggest that the twofactors: a) BMP-2 availability and b) RARα activity are important atdifferent stages during chondrogenesis. To further delineate the role ofBMP-2 and RARα in cartilage formation, we have used an approach thatinvolves incubation of micromass cultures with AGN194301 or BMP-2 fordifferent periods of time during culturing. To accomplish this BMP-2 orAGN194301 were added to cultures for the first 2 or 3 days then removed,or were added after 2 or 3 days of culturing. As has been shownpreviously (Roark and Greer (1994) Dev. Dynam. 200, 103-116), additionof BMP-2 at later culture periods yielded results that are comparable toexperiments in which BMP-2 is added continuously from the start ofculture (FIG. 5D-F). Conversely, addition of BMP-2 for the first 2 to 3days of culture caused an increase in nodule number but this increasewas not as dramatic as that observed upon adding BMP-2 later (i.e.,after 2 or 3 days). The average size of the nodules was also noticeablyincreased in cultures that were either continuously exposed to BMP-2 ortreated after 2 or 3 days of culture (FIG. 5B-F). These large nodulesradiate towards the outside of the culture and are, in part, aconsequence of the recruitment of proliferating uncommitted cellspresent in the periphery of the culture into the nodules. Similarobservations have been made with BMP-2 addition in vivo as Duprez et al.(Duprez et al. (1996) Mech. Dev. 57, 145-157) showed that expansion ofskeletal elements in the presence of BMP-2 or -4 was at the expense ofother cell populations in the developing chick limb bud. Hence, theconsequences of BMP-2 addition are much more pronounced in culturestreated at later stages of culture and likely reflect the ability ofBMP-2 to stimulate commitment of mesenchymal cells to the chondrocyticlineage with subsequent recruitment into nodules.

In contrast to BMP-2 and -4, AGN194301 had its most pronouncedstimulatory effect on nodule formation when it was present early in theculture period. When 1 μM AGN194301 was added to cultures for only thefirst 2 or 3 days, then removed, more cartilage nodules were formedcompared to untreated cultures (FIG. 5G-I) or to cultures exposed forlonger periods of time but untreated for the first 2 or 3 days (FIG. 5K,L). Interestingly, the increase in nodule number caused by earlytreatment of the antagonist was maintained for 4 or more days after itsremoval and was comparable to the increase in nodule number observedwith continuous treatment (FIG. 5H-J). AGN194301, therefore, caused anincrease in nodule formation, however the nodules were much smallercompared to cultures receiving antagonist at later culture times. WhenBMP-2 and AGN194301 were added together to micromass cultures at aconcentration of 10 ng/ml and 1 μM, respectively, there was a ˜100%increase in nodule number compared to untreated controls (FIG. 5M).BMP-2 addition alone to cultures initiated from the same limb budscaused a ˜45% increase, whereas AGN194301 treatment caused an increaseof ˜60% (FIG. 5M). Together, these results demonstrate that the loss ofRAR activity and the presence of a BMP signal are two events that act atdifferent stages of the chondrogenic sequence as they exhibit markedlydifferent chondrogenic stimulatory properties.

Example 6 Cartilage Formation is Rescued in Noggin-Treated Cultures byAddition of an RARα Antagonist

To further address the requirement for BMPs in chondrogenesis, wild-typelimb mesenchyme cultures were treated with Noggin, a secreted inhibitorof BMP-2, and -4. Addition of 10 ng/ml Noggin to wild-type culturesdramatically reduced cartilage nodule formation by ˜84% in comparison tountreated controls (FIG. 6 a, b and d). In an attempt to define thechondrogenic stage affected by Noggin, cultures treated with Noggin wereexamined for the expression of col II and col IIA an alternativelyspliced form of col II expressed in prechondrogenic cells (Sandell etal. 1991; Sandell et al. 1994). In limb bud derived cultures col IIA isexpressed in the condensing mesenchyme surrounding cartilaginous nodulesand in precartilaginous condensations (FIG. 6 e, f). If Noggin delays orinhibits chondroblast differentiation but does not affect condensation,then the decrease in col II expressing nodules should be accounted forby col IIA expressing condensations. In contrast, however, comparison ofexpression of these two genes by in situ hybridization in Noggin-treatedcultures indicates there are no additional condensations present (FIG. 6e, f). Hence, Noggin appears to interfere with formation ofprecartilaginous condensations.

Earlier experiments in which addition of BMP-2 was unable to rescuecartilage in transgene-expressing cultures coupled with the acceleratedappearance of col II expressing cells in antagonist-treated culturesdemonstrated that loss of RAR activity alone is sufficient to initiatethe chondrogenic differentiation program. To further demonstrate this,Noggin-treated cultures were exposed to the RAR antagonist. The additionof 1 μM AGN194301 to Noggin-treated cultures, stimulated cartilagenodule formation such that these cultures resembled untreated cultures(FIG. 6 a, c and d). In contrast, addition of 10 or 20 nM all-trans RAfurther diminished nodule formation in Noggin-treated cultures (data notshown). These results indicate that suppression of RAR-mediatedsignaling not only stimulates expression of the chondrogenic phenotype,but can do so independently of a BMP signal.

TABLE 1 AGN 194301

2-Fluoro-4-[(1-(8-bromo-2,2-dimethyl-4-(4-methylphenyl)-2-H-chromen-6-yl)-methanoyl)-amino]-benzoic acid

1-29. (canceled)
 30. A composition comprising: (a) an RAR antagonist;(b) a pharmaceutically acceptable carrier; and (c) a chondrogenicstimulator, wherein said composition induces chondrogenesis leading tocartilage formation or chondrogenesis leading to cartilage formationthat further mediates formation of new bone tissue in a vertebrate. 31.The composition according to claim 30, wherein said chondrogenicstimulator is a protein selected from the group consisting of a bonemorphogenetic protein (BMP), an osteogenic protein (OP), a cytokine andcombinations thereof.
 32. The composition according to claim 31, whereinsaid BMP is selected from the group consisting of BMP-2, BMP-4 andBMP-5.
 33. The composition according to claim 31, wherein saidosteogenic protein is OP-1.
 34. The composition according to claim 30,wherein the RAR antagonist is present in an amount for stimulatingchondrogenesis.
 35. The composition according to claim 30, wherein saidcomposition is provided as a solution, suspension, gel, matrix, cream,film, paste, capsule, pill or tablet, or is encapsulated withinliposomes.
 36. The composition according to claim 30, wherein saidcomposition is for intra-articular injection.
 37. The compositionaccording to claim 30, wherein said composition is provided within abiodegradable implantable matrix.
 38. The composition according to claim30, wherein said composition is for the treatment of arthritis, abnormalcartilage formation and/or cartilage defects.
 39. The compositionaccording to claim 30, wherein said RAR antagonist antagonizes one ormore of RARα, RARβ and RARγ.
 40. A device for implantation in avertebrate, comprising: (a) an implantable biocompatible carrier; and(b) a RAR antagonist dispersed within or on said carrier.
 41. The deviceaccording to claim 40, wherein said carrier comprises demineralizedbone, protein-extracted bone, particulate bone, allogenic bone,xenogenic bone or combinations thereof.
 42. The device according toclaim 40, wherein said carrier is selected from the group consisting ofsemi-permeable polymer matrices, hydroxyapatite, collagen, tricalciumphosphate and copolymers of glycolic, lactic and butyric acid.
 43. Thedevice according to claim 40, wherein said device comprises abiodegradable sponge.
 44. The device according to claim 40, wherein saiddevice is a prosthetic for repairing an orthopaedic defect, injury oranomaly in a vertebrate.
 45. The device according to claim 44, whereinsaid device has a surface region implantable adjacent or within a targettissue and said RAR antagonist is disposed on said surface region in anamount sufficient to promote enhanced chondrogenesis and associated bonetissue growth into said surface.
 46. The device according to of claim40, wherein said RAR antagonist is provided within a composition furthercomprising a pharmaceutically acceptable carrier and a chondrogenicstimulator.
 47. The device according to claim 46, wherein saidchondrogenic stimulator is a protein selected from the group consistingof a bone morphogenetic protein (BMP), an osteogenic protein (OP), acytokine and combinations thereof.
 48. The device according to claim 40,wherein said RAR antagonist antagonizes one or more of RARα, RARβ andRARγ.
 49. A method for producing a chondrocyte from a chondroprogenitormesenchymal cell, comprising contacting said chondroprogenitormesenchymal cell with an RAR antagonist or a RAR antagonist compositionaccording to claim 1 in vitro.
 50. The method according to claim 49,wherein said chondrocytes are implanted into a cartilage defect in vivoto produce cartilage at said site.
 51. The method according to claim 49,wherein said RAR antagonist antagonizes one or more of RARα, RARβ andRARγ. 52-56. (canceled)
 57. A method for stimulating chondroblastdifferentiation, comprising delivering an effective amount of a RARantagonist to inhibit RAR activity.
 58. The method of claim 57, whereinsaid RAR antagonist amount decreases transcriptional activation of theRA receptor.